Feline Myocardium Research Articles

Metabolic Markers of Insulin Resistance in Feline Hypertrophied Myocardium. Insulin Resistance in Heart Failure

Investigation of myocardial tissue concentration of ATP, glucose transporters 1 and 4, pyruvate dehydrogenase, hexokinase 2, insulin receptor and adropin proteins, determining metabolic changes and possible insulin resistance in feline myocardium with hypertrophic phenotype. This is the pilot study for metabolic markers determination. Eighteen cats were studied, divided into 3 groups: Without cardiac disease (n = 5), cats with hypertrophic cardiomyopathy (n = 8), cats with chronic kidney disease and secondary myocardial hypertrophy (n = 5). Animals in the study were diagnosed for the primary disease by standard methods and algorithms. Cats were euthanized due to the end-stage chronic kidney disease, refractory heart failure or by owners’ will. The myocardium samples were obtained immediately after death. Samples for the metabolic study were taken from the apical part of the left ventricular free wall and fixed in liquid nitrogen at once and were stored in -80°C refrigerator. Studied proteins concentrations were analyzed in a specialized research laboratory, using ELISA kits, provided by Cloud-Clone Corp. (USA), included: Total ATP, pyruvate dehydrogenase, hexokinase II, Adropin, insulin receptor, GLUT1 and GLUT4. In the group with HCM, we discovered that levels of ATP, pyruvate dehydrogenase and adropin were severely suppressed in comparison to healthy cats, while GLUT1 and GLUT4 did not change. The concentration of hexokinase 2 and insulin receptor proteins significantly increased. In the group of secondary myocardial hypertrophy, suppression of most studied proteins was admitted, except insulin receptor. In conclusion, we discovered metabolic remodeling and development of insulin resistance in observed diseases with hypertrophy phenotype. We observed depression of pivotal enzymes proteins, limiting energy restoration potency for cardio myocytes.

Feline Panleukopenia Virus Is Not Associated With Myocarditis or Endomyocardial Restrictive Cardiomyopathy in Cats.

Canine parvovirus-2 (CPV-2) is nearly indistinguishable from feline panleukopenia virus (FPV) and is a well-known cause of viral myocarditis in young puppies; however, it is not known whether either FPV or CPV-2 naturally infects feline cardiomyocytes and causes myocarditis. Endomyocarditis (EMC) and left ventricular endomyocardial fibrosis (LVEF), clinically known as "endomyocardial restrictive cardiomyopathy," are important feline heart diseases suspected to have an infectious etiology. A continuum is suggested with EMC representing the acute reaction to an unknown infectious agent and LVEF the chronic manifestation of repair. The purpose of this study was to determine (1) whether there is natural parvovirus infection of the feline myocardium and (2) whether parvoviral infection is associated with feline EMC and/or LVEF. In a retrospective study, polymerase chain reaction and sequencing for the parvovirus VP1/2 gene was performed on archived heart tissue from cats with endomyocardial disease and controls. Similar methods were used prospectively on myocardial tissues from shelter-source kittens. Although 8 of 36 (22%; 95% confidence interval [CI], 11%-40%) shelter kittens had parvoviral DNA in myocardial tissue, VP1/2 DNA was not detected in 33 adult cases or 34 controls (95% CI, 0% to ∼11%). These findings were confirmed by in situ hybridization: adult cats did not have detectable parvovirus DNA, although rare intranuclear signal was confirmed in 7 of 8 shelter-source kittens. In kittens, parvovirus was not significantly associated with myocarditis, and in situ hybridization signal did not colocalize with inflammation. Although infection of cardiomyocytes was demonstrated in kittens, these data do not support a role for parvovirus in EMC-LVEF.

Myocardial leptin transcription in feline hypertrophic cardiomyopathy

Leptin is an adipokine, which is in humans with cardiac disease suspected to be involved in myocardial remodeling and thrombus formation. In cats, however, it is not known whether leptin plays a role in cardiac disease, i.e. hypertrophic cardiomyopathy (HCM) and the presence of an atrial thrombus (AT). The objective of the study was therefore to establish whether leptin is transcribed in the feline myocardium and to compare myocardial leptin mRNA concentrations in cats with HCM with and without AT, and in cats without cardiac diseases. Myocardial samples from 15 cats with HCM (five of these with AT), and 12 cats without cardiac diseases were investigated for leptin mRNA expression using quantitative reverse transcriptase PCR, and the transcription levels were correlated with those obtained for a range of cytokines and remodeling parameters.Leptin mRNA expression was detected in the myocardium in all heart regions, with generally higher concentrations in the atria than in the ventricles. Cats with HCM exhibited higher atria and ventricular leptin transcription than cats without cardiac diseases, but reduced ventricular transcription levels in the presence of AT. A positive correlation between leptin, cytokine and remodeling marker transcription levels was observed.The present study shows that leptin is constitutively transcribed in the feline myocardium. The observed increase in leptin mRNA concentrations in the myocardium from cats with HCM and the reduction when an AT is present suggests varying gene activation in different stages of the disease and a potential involvement of leptin in the feline cardiac remodeling process.

An Immunohistochemical Study of Feline Myocardial Fibrosis

The aim of the present study was to investigate the pathology of feline myocardial fibrosis. The hearts from 40 cats with myocardial fibrosis were compared with the hearts from 25 normal cats. Clinical data were available in 11 cases. Hearts with myocardial fibrosis were hypomotile and there were hyperechoic areas in the ventricular wall on echocardiography. The presence of myocardial fibrosis was correlated significantly with hypertrophy of the ventricles, atrial dilation and angiosclerosis. Immunohistochemical studies demonstrated that normal feline cardiomyocytes expressed matrix metalloproteinase (MMP)-2, MMP-9, MMP-14, tissue inhibitor of matrix metalloproteinase (TIMP)-2 and transforming growth factor (TGF)-β2. Fibroblasts in normal hearts expressed only TIMP-2. In the hearts with myocardial fibrosis, expression of MMP-2, TIMP-3 and TGF-β2 by cardiomyocytes was significantly increased, but TIMP-2 expression was diminished. Fibroblasts in the affected hearts showed expression of MMP-14 in several cases. These findings suggest that a complex fibrotic remodelling of the feline myocardium occurs in this disease and that cardiomyocytes are involved in this process.

Site-specific Microtubule-associated Protein 4 Dephosphorylation Causes Microtubule Network Densification in Pressure Overload Cardiac Hypertrophy

In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac alpha- and beta-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 --> Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality.

In vivo administration of calpeptin attenuates calpain activation and cardiomyocyte loss in pressure-overloaded feline myocardium.

Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv) by bolus injections twice, 15 min before and 6 h after induction of 24-h PO. Calpeptin blocked the following PO-induced changes: calpain enrichment and activation, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone H2B phosphorylation. Although similar administration of a caspase inhibitor, N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VD-fmk), blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei, are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy.

Mitochondrial reactive oxygen species activate the slow force response to stretch in feline myocardium

When the length of the myocardium is increased, a biphasic response to stretch occurs involving an initial rapid increase in force followed by a delayed slow increase called the slow force response (SFR). Confirming previous findings involving angiotensin II in the SFR, it was blunted by AT1 receptor blockade (losartan). The SFR was accompanied by an increase in reactive oxygen species (ROS) of approximately 30% and in intracellular Na(+) concentration ([Na(+)](i)) of approximately 2.5 mmol l(-1) over basal detected by H(2)DCFDA and SBFI fluorescence, respectively. Abolition of ROS by 2-mercapto-propionyl-glycine (MPG) and EUK8 suppressed the increase in [Na(+)](i) and the SFR, which were also blunted by Na(+)/H(+) exchanger (NHE-1) inhibition (HOE642). NADPH oxidase inhibition (apocynin or DPI) or blockade of the ATP-sensitive mitochondrial potassium channels (5HD or glybenclamide) suppressed both the SFR and the increase in [Na(+)](i) after stretch, suggesting that endogenous angiotensin II activated NADPH oxidase leading to ROS release by the ATP-sensitive mitochondrial potassium channels, which promoted NHE-1 activation. Supporting the notion of ROS-mediated NHE-1 activation, stretch increased the ERK1/2 and p90rsk kinases phosphorylation, effect that was cancelled by losartan. In agreement, the SFR was cancelled by inhibiting the ERK1/2 signalling pathway with PD98059. Angiotensin II at a dose that mimics the SFR (1 nmol l(-1)) induced an increase in .O(2)(-) production of approximately 30-40% detected by lucigenin in cardiac slices, an effect that was blunted by losartan, MPG, apocynin, 5HD and glybenclamide. Taken together the data suggest a pivotal role of mitochondrial ROS in the genesis of the SFR to stretch.

Reduced sarcoplasmic reticulum Ca 2+ load mediates impaired contractile reserve in right ventricular pressure overload

Myocardial contractile reserve is significantly attenuated in patients with advanced heart failure. The aim of this study was to identify mechanisms of impaired contractile reserve in a large animal model that closely mimics human myocardial failure. Progressive right ventricular hypertrophy and failure were induced by banding the pulmonary artery in kittens. Isometric contractile force was measured in right ventricular trabeculae ( n = 115) from age-matched Control and Banded feline hearts. Rapid cooling contractures (RCC) were used to determine sarcoplasmic reticulum (SR) Ca 2+ load while assessing the ability of changes in rate, adrenergic stimulation and bath Ca 2+ to augment contractility. The positive force–frequency relationship and robust pre- and post-receptor adrenergic responses observed in Control trabeculae were closely paralleled by increases in RCC amplitude and the RCC2/RCC1 ratio. Conversely, the severely blunted force–frequency and adrenergic responses in Banded trabeculae were paralleled by an unchanged RCC amplitude and RCC2/RCC1 ratio. Likewise, supraphysiologic levels of bath Ca 2+ were associated with severely reduced contractility and RCC amplitude in Banded trabeculae compared to Controls. There were no differences in myofilament Ca 2+ sensitivity or length-dependent increases in contractility between Control and Banded trabeculae. There was a significant decrease in SR Ca 2+-ATPase pump abundance and phosphorylation of phospholamban and ryanodine receptor in Banded trabeculae compared with Controls. A reduced ability to increase SR Ca 2+ load is the primary mechanism of reduced contractile reserve in failing feline myocardium. The similarity of impaired contractile reserve phenomenology in this feline model and transplanted hearts suggests mechanistic relevance to human myocardial failure.

Contractile Reserve in Normal and Failing Feline Myocardium: Importance of Sarcoplasmic Reticulum Ca 2+ Load and Na +/Ca 2+ Exchanger Activity

Contractile Reserve in Normal and Failing Feline Myocardium: Importance of Sarcoplasmic Reticulum Ca 2+ Load and Na +/Ca 2+ Exchanger Activity

Arginase: a modulator of myocardial function

when nitric oxide (NO) donors were first administered to isolated, normal cardiomyocytes, a negative, cGMP-dependent inotropic effect was observed, whereas inhibitors of NO synthases (NOS) had no effect. Further studies then demonstrated a biphasic contractile response to exogenous NO and cGMP,

Modulation of contractility by myocyte-derived arginase in normal and hypertrophied feline myocardium

L-Arginine, the sole substrate for the nitric oxide (NO) synthase (NOS) enzyme in producing NO, is also a substrate for arginase. We examined normal feline hearts and hearts with compensated left ventricular (LV) hypertrophy (LVH) produced by ascending aorta banding. Using Western blot analysis, we examined the abundance of arginase isozymes in crude homogenates and isolated cardiac myocytes obtained from the LVs of normal and LVH hearts. We examined the functional significance of myocyte arginase via measurement of shortening and intracellular calcium in isolated myocytes in the presence and absence of boronoethyl chloride (BEC), a specific pharmacological inhibitor of arginase. Both arginase I and II were detected in crude myocardial homogenates, but only arginase I was present in isolated cardiac myocytes. Arginase I was downregulated in LVH compared with normal. Inhibition of arginase with BEC reduced fractional shortening, maximal rate of shortening (+dL/dt) and relengthening (-dL/dt), and the peak of the free cytosolic calcium transient in normal myocytes but did not affect these parameters in LVH myocytes. These negative inotropic actions of arginase inhibition were associated with increases in cGMP generation. These studies indicate that only arginase I is present in cardiac myocytes where it tends to limit NO and cGMP production with the effect of supporting basal contractility. In experimental LVH induced by pressure overload, our studies demonstrate reduced arginase I expression and reduced functional significance, allowing greater arginine substrate availability for NO/cGMP signaling.

Relations between ?-adrenoceptor occupancy and increases of contractile force and adenylate cyclase activity induced by catecholamines in human ventricular myocardium

The function of beta-adrenoceptors was investigated in ventricular myocardium obtained from patients undergoing open heart surgery. 1. Dopamine increased contractile force up to 1/2 and 1/4 of the maximum increase caused by (-)-noradrenaline or (-)-adrenaline in right and left ventricular preparations, respectively. 2. beta-Adrenoceptors were labelled with 3H-(-)-bupranolol. For 3/4 of the receptors (beta 1) the affinity of (-)-noradrenaline was 20 times higher than for the remaining 1/4 (beta 2). (-)-Adrenaline and dopamine appeared to be non-selective for beta 1 and beta 2. 3. Dopamine was able to stimulate the adenylate cyclase only up to 1/3 of the maximum stimulation caused by (-)-noradrenaline and (-)-adrenaline. 4. Increases in contractile force by (-)-noradrenaline were closely associated with small increases of cyclase activity through beta 1-adrenoceptors, consistent with a common link. 5. The experiments on human myocardium were compared with similar experiments on feline myocardium. Feline ventricle exhibited a 20- to 30-fold higher sensitivity to catecholamines as activators of contractile force than did human ventricle. However, the binding affinities for catecholamines were similar in cat and man. 6. A 3 h exposure of human and feline ventricular myocardium to (-)-isoprenaline caused desensitization by uncoupling beta-adrenoceptors from the adenylate cyclase. Desensitization reduced the maximum contractile response to (-)-isoprenaline in human but not in feline ventricle. 7. The more efficient activation of contractile force by (-)-noradrenaline in cat, compared to man, appears to be related to a 2-fold higher density of beta 1-adrenoceptors, a 6-fold higher production of cyclic AMP per beta 1-adrenoceptor and possibly to a more effective use of cyclic AMP for contraction.

Positive Inotropic and Negative Lusitropic Effect of Angiotensin II: Intracellular Mechanisms and Second Messengers

In the cat ventricle angiotensin II exerts a positive inotropic effect produced by an increase in intracellular calcium associated with a prolongation of relaxation. The signaling cascades involved in these effects as well as the subcellular mechanisms of the negative lusitropic effect are still not clearly defined. The present study was directed to investigate these issues in cat papillary muscles and isolated myocytes. The functional suppression of the sarcoplasmic reticulum (SR) with either 0.5 μ m ryanodine or 0.5 μ m ryanodine plus 1 μ m thapsigargin or the preincubation of the myocytes with the specific inhibitor of the inositol 1,4,5-triphosphate (IP3) receptors [diphenylborinic acid, ethanolamine ester (2-APB), 5–50 μ m] did not prevent the positive inotropic effect and the increment in Ca2+transient produced by 1 μ m angiotensin II. In contrast, protein kinase C (PKC) inhibitors, chelerythrine (20 μ m) and calphostin C (1 μ m) completely inhibited both, the angiotensin II-induced increase in L-type calcium current and positive inotropic effect. The prolongation of half relaxation time produced by 0.5 μ m angiotensin II [207±15.4 msec (control) to 235±19.98 msec (angiotensin II),P <0.05] was completely blunted by PKC inhibition. This antirelaxant effect, which was independent of intracellular pH changes, was associated with a prolongation of the action potential duration and was preserved after either the inhibition of the SR and the SR Ca2+ATPase (ryanodine plus thapsigargin) or of the reverse mode of the Na+/Ca2+exchanger (KB-R7943, 5 μ m). We conclude that in feline myocardium the positive inotropic and negative lusitropic effects of angiotensin II are both entirely mediated by PKC without any significant participation of the IP3limb of the phosphatidylinositol/phospholipase C cascade. The results suggest that the antirelaxant effect of angiotensin II might be determined by the decrease in Ca2+efflux through the Na+/Ca2+exchanger produced by the angiotensin II-induced prolongation of the action potential duration.

Integrin Activation and Focal Complex Formation in Cardiac Hypertrophy

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

Hemodynamic regulation of tumor necrosis factor-alpha gene and protein expression in adult feline myocardium.

Tumor necrosis factor-alpha (TNF-alpha) mRNA and protein biosynthesis were examined in adult feline myocardium in the presence and absence of superimposed hemodynamic pressure overloading. A brief period of hemodynamic pressure overloading ex vivo resulted in de novo TNF-alpha mRNA expression within 30 minutes and de novo TNF-alpha protein production within 60 minutes; neither TNF-alpha mRNA nor protein was detected in hearts perfused at normal perfusion pressures. Moreover, TNF-alpha mRNA and protein biosynthesis were observed in myocyte and nonmyocyte cell types in the pressure-overloaded hearts. To determine whether a simple passive stretch of the myocardium was a sufficient stimulus for TNF-alpha biosynthesis, we examined TNF-alpha mRNA expression in stretched and unstretched papillary muscles. This study showed that myocardial stretch was a sufficient stimulus for the induction of TNF-alpha mRNA biosynthesis. The functional significance of the intramyocardial production of TNF-alpha was determined by examining cell motion in isolated contracting cardiac myocytes treated with superfusates from pressure-overloaded and control hearts. These studies showed that cell motion was depressed in myocytes treated with superfusates from the pressure-overloaded hearts but was normal with the superfusates from the control hearts. Finally, hemodynamic pressure overloading in vivo under physiological conditions was also shown to result in de novo intramyocardial TNF-alpha mRNA biosynthesis. In conclusion, this study constitutes the initial demonstration that the adult mammalian myocardium elaborates biologically active TNF-alpha, both ex vivo and in vivo, in response to hemodynamic pressure overloading.

Dissociation between positive inotropic and alkalinizing effects of angiotensin II in feline myocardium

The present study examines the intracellular pH (pHi) dependence of angiotensin (ANG) II-induced positive inotropic effect in cat papillary muscles contracting isometrically (0.2 Hz, 30 degrees C). Muscles were loaded with the fluorescent dye 2'-7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester for simultaneous measurement of pHi and contractility. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (n = 4), there was a temporal dissociation between the positive inotropic and the alkalinizing effects of ANG II (0.5 microM). The positive inotropic effect of ANG II peaked at 9.7 +/- 0.8 min (240 +/- 57% above control) without significant changes in pHi. The increase in pHi became significant (0.05 +/- 0.01 pH units) only after 16 min of exposure to the drug, when the positive inotropic effect of ANG II was already fading. In HCO3- buffer (n = 7), the ANG II-induced positive inotropic effect occurred without significant pHi changes. In the presence of 5 microM ethyl isopropyl amiloride (EIPA, to specifically inhibit the Na+/H+ exchanger), the alkalinizing effect of ANG II was changed to a significant decrease in pHi, despite which ANG II still increased contractility by 87 +/- 16% (n = 6). The results indicate that in HEPES buffer only a fraction of the ANG II-induced positive inotropic effect can be attributed to a pHi change, whereas in a physiological CO2-HCO3- medium the positive inotropic effect of ANG II is independent of pHi changes. Furthermore, an ANG II-induced increase in myocardial contractility was observed even when ANG II administration elicited a decrease in pHi, as occurred after Na+/H+ exchanger blockade. The results show that in feline myocardium, the increase in contractility evoked by ANG II in a physiological CO2-HCO3- medium is not due to an increase in Ca2+ myofilament sensitivity secondary to an increase in myocardial pHi.

Carvedilol improves function and reduces infarct size in the feline myocardium by protecting against lethal reperfusion injury

This study examined the effect of carvedilol, a vasodilating β-adrenoceptor antagonist and antioxidant, on lethal reperfusion injury in feline hearts subjected to 40 min of regional ischaemia and 180 min of reperfusion. 30 open chest anaesthetized cats were randomized into three groups. A control group ( n = 10) was compared with a group given carvedilol before coronary artery occlusion ( n = 10) and a group given carvedilol immediately before and during early reperfusion ( n = 10). Regional myocardial function was measured by sonomicrometry. Infarct size was determined by staining the left ventricle with triphenyl tetrazolium chloride. Myocardial blood flow was measured by radiolabeled microspheres. Tissue levels of glutathione were measured after reperfusion. Infarct size was significantly reduced compared to control both when carvedilol was given before ischaemia (0.2 ± 0.1 vs. 17.6 ± 3.6%, P < 0.05) and when given immediately before reperfusion (3.7 ± 1.3 vs. 17.6 ± 3.6%, P < 0.05). Regional shortening improved significantly and the incidence of ventricular fibrillation during early reperfusion was reduced in both groups treated with carvedilol compared to control. Oxidized glutathione did not differ between groups in the post-ischaemic myocardium. This study supports that lethal reperfusion injury is a significant phenomenon. Furthermore, carvedilol reduces infarct size and reperfusion arrhythmias, and improves post-ischaemic regional myocardial function by protecting against both ischaemic and lethal reperfusion injury. The present study does not answer whether it is the non-selective β- or α 1-adrenoceptor antagonism, the antarrhytmic or the antioxidant actions of carvedilol that is responsible for the protective effect.

Ventricular arrhythmogenic dose of adrenaline during sevoflurane, isoflurane, and halothane anaesthesia either with or without ketamine or thiopentone in cats

The doses of adrenaline required to induce ventricular arrhythmia during sevoflurane, isoflurane and halothane anaesthesia, either with or without infusions of ketamine (76 μg kg −1 min −1) or thiopentone (0·5 mg kg −1 min −1), were determined in cats. Groups of six to eight cats were maintained at end-tidal concentrations equivalent to 1·25 times the minimal alveolar concentration of each anaesthetic. The mean dose of adrenaline required to induce arrhythmia during sevoflurane anaesthesia (19·0 μg kg −1) was approximately 11 times higher than that required during halothane anaesthesia (1·66 μg kg −1) and the same as that required during isoflurane anaesthesia (19·0 μg kg −1). Ketamine tended to decrease the requirement of adrenaline during halothane anaesthesia, but not significantly, and did not change the requirement during isoflurane or sevofturane anaesthesia. Thiopentone did not change the requirement for adrenaline during halothane, isoflurane or sevoflurane anaesthesia. It was concluded that either with or without ketamine or thiopentone, the effect of sevoflurane on the sensitisation of the feline myocardium to the arrhythmogenic effects of adrenaline was significantly less than that of halothane and not different that of isoflurane.

Tumor necrosis factor-alpha gene and protein expression in adult feline myocardium after endotoxin administration.

TNF alpha mRNA and protein biosynthesis were examined in the adult feline heart after stimulation with endotoxin. When freshly isolated hearts were stimulated with endotoxin in vitro, de novo TNF alpha mRNA expression occurred within 30 min, and TNF alpha protein production was detected within 60-75 min; however, TNF alpha mRNA and protein production were not detected in diluent-treated hearts. Immunohistochemical studies localized TNF alpha to endothelial cells, smooth muscle cells, and cardiac myocytes in the endotoxin-treated hearts, whereas TNF alpha immunostaining was absent in the diluent-treated hearts. To determine whether the cardiac myocyte was a source for TNF alpha production, two studies were performed. First, in situ hybridization studies, using highly specific biotinylated probes, demonstrated TNF alpha mRNA in cardiac myocytes from endotoxin-stimulated hearts; in contrast, TNF alpha mRNA was not expressed in myocytes from diluent-treated hearts. Second, TNF alpha protein production was observed when cultured cardiac myocytes were stimulated with endotoxin, whereas TNF alpha protein production was not detected in the diluent-treated cells. The functional significance of the intramyocardial production of TNF alpha was determined by examining cell motion in isolated cardiac myocytes treated with superfusates from endotoxin- and diluent-stimulated hearts. These studies showed that cell motion was depressed in myocytes treated with superfusates from the endotoxin-treated hearts, but was normal with the superfusates from the diluent-treated hearts; moreover, the negative inotropic effects of the superfusates from the endotoxin-treated hearts could be abrogated completely by pretreatment with an anti-TNF alpha antibody. Finally, endotoxin stimulation was also shown to result in the intramyocardial production of TNF alpha mRNA and protein in vivo. Thus, this study shows for the first time that the adult mammalian myocardium synthesizes biologically active TNF alpha.

The physiological and anatomical demonstration of functionally selective parasympathetic ganglia located in discrete fat pads on the feline myocardium

Experiments utilizing surgical parasympathectomy of discrete fat pad ganglia on the surface of the heart have suggested that there are two anatomically segregated and physiologically independent parasympathetic intracardiac ganglia which are capable of selective control of sino-atrial (SA) rate and atrio-ventricular (AV) conduction. Some pharmacological data, however, are inconsistent with these conclusions. We have examined the cardiodynamic effects of discrete injections of a ganglionic blocking drug into two fat pads on the surface of the cat heart. These fat pads were shown to contain ganglion cells histologically. It was observed that vagal effects upon cardiac rate are selectively mediated by neurons located in ganglia overlying the right pulmonary veins at the junction of the right atrium and superior vena cava. On the other hand, vagal effects upon AV conduction were selectively mediated by neurons located in a fat pad at the junction of the inferior vena cava and the inferior left atrium. These pharmacological data support the concept that specific intracardiac ganglia are capable of selective control of SA rate and AV conduction.